Enhancers are a class of cis-acting regulatory sequences that can activate transcription in a directionally-independent manner over long distances. Although first identified in viral genomes, they have been implicated in the cell-type or tissue-specific control of an increasing number of cellular genes. Accumulating evidence suggests that the action of enhancers involves their interaction with specific protein factors that can confer both positive and negative regulation of enhancer activity. The objectives of this research will be to identify and characterize protein factors which interact with the enhancer of an avian retrovirus, Rous Sarcoma Virus (RSV). This enhancer is constiutively active in essentially all cell types, and it is anticipated that an understanding of the molecular mechanisms responsible for its activation of transcription will be generally applicable to other enhancers. Recently, using sensitive assays designed to identify sequence-specific DNA binding proteins in crude nuclear extracts, at least two proteins (EFI, EFII) have been detected in avian cell extracts that specifically bind to different nucleotide sequences in the RSV LTR enhancer. Site-directed mutagenesis and the construction of synthetic enhancers will be employed to further evaluate the functional significance of EFI and EFII (and possible additional RSV enhancer factors) in mediating LTR enhancer activity in vivo. Purification of these factors by newly developed affinity chromatography methods will permit their identification and further characterization. Specific plans include attempting to reproduce RSV LTR enhancer in a soluble transcription system in vitro so that the mechanisms by which these proteins act can be further evaluated. We will also exploit recently developed in vitro chromatin assembly systems to evaluate the influence of RSV LTR enhancer factors on chromatin organization and transcription in vitro. Finally, we will attempt to obtain cDNA clones for EFI and EFII so that we can establish their primary structure and utilize site-specific mutagenesis to establish structural and functional relationships. Acquiring enhancer factor cDNA clones will also enable these proteins to be overexpressed in bacteria, potentially providing large quantities of material for biochemical and biophysical studies of an important class of regulatory proteins that would otherwise be unfeasible.